Microdomains, called lipid rafts, exist in all mammalian cell membranes. They are rich in cholesterol and sphingolipids, and play important roles in various cellular processes including signal transduction, cell surface polarity, and endocytosis (Simons and Ikonen (2000) Science 290:1721-6). Cholesterol also modulates intracellular transport of proteins from early endosomes to the plasma membranes, or from endosomes to the Golgi (Mayor, et al. (1998) EMBO J. 17:4626-38; Grimmer, et al. (2000) Mol. Biol. Cell 11:4205-16; Miwako, et al. (2001) J. Cell Sci. 114:1765-76) and the trafficking pathway of other lipids such as sphingolipids (Puri, et al. (1999) Nat. Cell Biol. 1:386-8; Puri, et al. (2001) J. Cell Biol. 154:535-47). One of the key molecules involved in the correct distribution of intracellular cholesterol is Niemann-Pick Type C1 (NPC1) protein; mutation in NPC1 causes NPC syndrome, a fatal pediatric neurodegenerative disease (Patterson, et al. (2001) In: The Metabolic and Molecular Bases of Inherited Disease, Scriver, et al. (ed) McGraw-Hill, New York p. 3611-3633). Chinese hamster ovary (CHO) mutants that are defective in the NPC1 protein have been isolated and characterized (Cadigan, et al. (1990) J. Cell Biol. 110:295-308; Gu, et al. (1997) Proc. Natl. Acad. Sci. USA 94:7378-83; Dahl, et al. (1992) J. Biol. Chem. 267:4889-96). Cholesterol trafficking activities of NPC1 mutants (CT43 and CT60) and their parental cell line 25RA have been shown (Chang and Limanek (1980) J. Biol. Chem. 255:7787-95; Hua, et al. (1996) Cell 87:415-26). NPC1 is involved in the transport of low-density lipoprotein (LDL)-derived cholesterol from internal compartments to the plasma membrane or to the ER (Cruz and Chang (2000) J. Biol. Chem. 275:41309-16; Pentchev, et al. (1985) Proc. Natl. Acad. Sci. USA 82:8247-51; Liscum, et al. (1989) J. Cell Biol. 108:1625-1636). NPC1 also participates in the transport of plasma membrane-derived cholesterol and endogenously synthesized cholesterol to the ER (Cruz and Chang (2000) supra; Cruz, et al. (2000) J. Biol. Chem. 275:4013-21; Pentchev, et al. (1985) supra; Byers, et al. (1992) Biochim. Biophys. Acta 1138:20-6; Lange, et al. (2000) J. Biol. Chem. 275:17468-75). Irrespective of the origin of cholesterol, the lack of a functional NPC1 protein invariably leads to cholesterol accumulation in the late endosome/lysosome.
Monitoring intracellular cholesterol transport using a biochemical approach is problematic as the isolation of distinct subcellular organelles in their pure states is difficult. Microscopic approaches have provided invaluable information. A well-known method for specifically detecting cholesterol in intact cells uses filipin, a naturally fluorescent polyene antibiotic that has high affinity towards cholesterol (Norman, et al. (1972) J. Biol. Chem. 247:1918-29), however, problems exist (Miller (1984) Cell Biol. Int. Rep. 8:519-35; Robinson and Karnovsky (1980) J. Histochem. Cytochem. 28:161-8; Severs and Simons (1983) Nature 303:637-8; Behnke, et al. (1984) Eur. J. Cell Biol. 35:200-15; Pelletier and Vitale (1994) J. Histochem. Cytochem. 42:1539-54). For example, the absorption spectrum of filipin is within the ultraviolet (UV) range and most of the commercially available confocal microscopes are not equipped with the laser beam that excites at the UV range; the fluorescence signal of filipin bleaches in a short time; in unfixed or fixed cells, filipin deforms the cellular membrane by forming complexes with cholesterol and causes perturbation of membrane lipid organization; and filipin has been reported to give false-negative results. Two fluorescent analogs of cholesterol, NBD-cholesterol and dehydroergosterol (DHE), have been used to track the fate of free sterol in the cell (Frolov, et al. (2000) J. Biol. Chem. 275:12769-80; Mukherjee, et al. (1998) Biophys. J. 75:1915-25). NBD-cholesterol exhibits a strong initial fluorescence signal at desirable wavelengths but bleaches quickly upon light exposure. Moreover, NBD-cholesterol does not consistently mimic the behavior of cholesterol inside the cells (Frolov, et al. (2000) supra). DHE behaves in a manner similar to cholesterol in many ways (Mukherjee, et al. (1998) supra). However, the fluorescence intensity of DHE is weak and it absorbs and emits in the UV region, therefore, special equipment is required in order to visualize DHE by fluorescence microscopy.
BCθ was developed as an effective tool for detecting cholesterol-rich domains (Waheed, et al. (2001) Proc. Natl. Acad. Sci. USA 98:4926-31; Iwamoto, et al. (1997) Biochim. Biophys. Acta 1327:222-30). BCθ is derived from a pore-forming cytolysin produced by the pathogenic bacterium Clostridium perfringens (Iwamoto, et al. (1997) supra). This thiol-activated cytolysin, called θ-toxin (or perfringolysin O), specifically binds to free (unesterified) cholesterol (Ohno-Iwashita, et al. (1990) Biochim. Biophys. Acta 1023:441-8; Ohno-Iwashita, et al. (1991) J. Biochem. (Tokyo) 110:369-75; Ohno-Iwashita, et al. (1992) Biochim. Biophys. Acta 1109:81-90; Nakamura, et al. (1995) Biochemistry 34:6513-20) and forms oligomeric pores in the membranes (Rossjohn, et al. (1997) Cell 89:685-92). BCθ is prepared by a two-step procedure. First, θ-toxin is proteolytically digested with subtilisin Carlsberg; this step generates a complex of 38- and 15-kDa fragments called Cθ (Ohno-Iwashita et al. (1986) Biochemistry 25:6048-53). Subsequently, the Cθ complex is biotinylated and purified, providing BCθ complex (Iwamoto, et al. (1997) supra). The biotinylation allows Cθ identification with avidin or streptavidin. When used in fluorescence microscopy, the labeling efficiency of BCθ depends on the qualities of fluorescent avidin and streptavidin. BCθ binds to cholesterol in synthetic liposomes and in intact cells with affinity identical to that of the wild-type θ-toxin, but because it does not oligomerize, it bears no hemolytic activity (Iwamoto, et al. (1997) supra; Ohno-Iwashita, et al. (1991) supra; Ohno-Iwashita, et al. (1992) supra). BCθ binds to cholesterol-rich microdomains in the plasma membrane of intact cells with or without fixation (Waheed, et al. (2001) supra; Hagiwara, et al. (1999) Biochem. Biophys. Res. Commun. 260:516-21; Möbius, et al. (2002) J. Histochem. Cytochem. 50:43-55); however, a means of detecting intracellular cholesterol using BCθ has not been demonstrated.
Accordingly, there is a need for a method of reliably and specifically detecting cholesterol for microscopic studies of intracellular cholesterol movement and accumulation.